Identification and characterization of a broad range of non-covalent interactions between target molecules and their ligands, such as the biospecific interaction of proteins, or the interaction between a drugs and a particular binder protein,—is with growing significance in biochemistry research and drug discovery. Protein-protein interactions e.g. at a cellular level play a major role in signal transduction events, and consequently in cellular response to external stimuli and the pathophysiological alteration of diseased cells. Conventional technologies for a specific, direct detection of such non-covalent complexes include Western blot, whereby the protein extract is separated by gel electrophoresis, (Kemeny, D. M. and Challacombe, S. J. (eds.), in “ELISA and other Solid Phase Immunoassays”; John Wiley & Sons, Chichester (1988)), ELISA and Radioligand Binding Assays (Berson et al., Clin. Chim. Acta, 22:51-60 (1968); Chard, T. “An Introduction to Radioimmunoassay and Related Techniques,” Elsevier Biomedical Press, Amsterdam, p 95-110 (1982)), Surface-Plasmon Resonance (Karlsson et al., J. Immunol. Methods, 145:229-240 (1991); Jonsson et al., Biotechniques, 11(5):620-627 (1991)), or Scintillation Proximity Assays (Udenfriend et al, Anal. Biochem., 161:494-500 (1987)). The radioligand binding assays are typically useful only when assessing the competitive binding of the unknown at the binding site for that of the radioligand and also require the use of radioactivity. The surface-plasmon resonance technique is a useful technology for measuring binding kinetics, and dissociation and association constants derived from such measurement are also helpful in elucidating the nature of the target-ligand interactions. However, all of these assays typically involve laborious and time-consuming experimentation or have to be performed by specifically educated individuals. All of these assays also require the user to know either the target molecule or the ligand prior to analysis because each require some type of preparation (i.e. label or immobilization) of the target or ligand molecule.
In recent years applications of mass spectrometry in the biosciences have been reported (Meth. Enzymol., Vol. 193, Mass Spectrometry (McCloskey, ed.; Academic Press, NY 1990); McLaffery et al., Acc. Chem. Res. 27:297-386 (1994); Chait and Kent, Science 257:1885-1894 (1992); Siuzdak, Proc. Natl. Acad. Sci., USA 91:11290-11297 (1994)), including methods for mass spectrometric analysis of biopolymers (Hillenkamp et al. (1991) Anal. Chem. 63:1193A-1202A; US 2004/0229369A1;U.S. Pat. No. 6,558,902). The so-called “soft ionization” mass spectrometric methods, including Matrix-Assisted Laser Desorption/Ionization (MALDI) and ElectroSpray Ionization (ESI), allow intact ionization, detection and mass determination of large molecules, i.e., well exceeding 300 kDa in mass (Fenn et al., Science 246:64-71 (1989); Karas and Hillenkamp, Anal. Chem. 60:2299-3001 (1988)).
Both MALDI mass spectrometry (MALDI-MS; reviewed in Nordhoff et al., Mass Spectrom. Rev. 15:67-138 (1997)) and ESI-MS have been used to analyze non-covalent protein complexes (Cohen et al, J. Am. Soc. Mass Spectrom. 8:1046-1052 (1997); Rosinke et al, J. Mass. Spec 30:1462-1468 (1995); Schar, M. Chimia 51:782-785 (1997); Woods et al. Anal. Chem. 67:4462-4465 (1995); Tito et al, Biophysical Journal 81:3503-3509 (2001); U.S. Pat. No. 6,329,146). Yet, for the study of undigested, unfragmented protein complexes by mass spectrometry, the sample preparation protocols and the instrument setup need to be adapted for each protein complexes targeted. Finding the favourable conditions to observe intact ions from protein complexes is time consuming and still a major difficulty to making these measurements routine in order to bring these studies from one-at-a-time to higher throughput. For MALDI MS, in order to detect non-covalent complexes special conditions, such as matrices solutions without organic solvent or soft laser analysis (i.e. first shot analysis) have to be determined. (Farmer and Caprioli, J. Mass Spectrom., (1998); Zehl and Allmaier, Rapid Commun. Mass Spectrom., (2003); Cohen, et al., JASMS, (1997)) Furthermore, most of these studies suffer from low signal intensities compared to those of the individual component due to instability of these non-covalent complexes and ease of dissociation during sample preparation and complex ionization.
There are very limited studies on intact undigested, unfragmented complexes by mass spectrometry. Furthermore, the study of crosslinked intact protein complexes is restricted to the analysis of homo-multimeric complex. These particular complexes naturally minimize analytical problems related with ion detection using MALDI MS, such as competition for ionization in the desorption plume or detector saturation decreasing detection of the complexes because of low mass ions. Yet, such homo-multimeric protein complexes are still a biological exception and the method has not been applied to other relevant biological complexes (T. B. Farmer, R. M. Caprioli, Biol Mass Spectrom 20, 796 (December, 1991)).
On the other hand, the study of digested protein complexes is an efficient yet indirect way to detect non-covalent protein complexes (Parker, C. E. and Tomer K. B, Molecular Biotechnology 20:49-62 (2002); WO 2002/058533A2). The method, is restricted by the selective desorption/ionization phenomenon and by the overlapping phenomenon due to the complexity of the peptide mixture obtained after proteolysis of the protein complexes. Also, because the spectra are an analysis of many peptide fragments of the complex it often takes complicated computer software in order to interpret the spectra. Sometimes in order to aid in identification of the peptide fragments, molecular tags are linked to the complex (US 2005/0095654A1; EP 0 850 320 B1; EP 1 150 120 A2; U.S. Pat. No. 6,635,452)
Clearly although the techniques referenced above show some degree of utility in performing certain types of general analyses, shortcomings in high sensitivity, ease of operation or understanding, and analytical compatibility remain.
Thus, there is a need for the development of robust, accurate, sensitive and reliable methods to routinely detect and analyze intact ions from undigested, unfragmented supramolecular target-ligand-complexes from both purified multicomponent samples or heterogeneous biological matrices, which will overcome the above shortcomings.
Applicants have now discovered methods to analyse the presence or the identity of intact ions of undigested, unfragmented supramolecular target-ligand-complexes from either purified multicomponent mixtures or heterogeneous biological matrices with high sensitivity and accuracy using mass spectrometry, in particular MALDI ToF mass spectrometry using sensitive high mass detection for a robust and routine analysis.
In particular the methods of the present application allow the analysis of intact non-covalent interactions between a target molecule and its ligand with high sensitivity and accuracy by first crosslinking the non-covalently bound target-ligand-complex and subsequently subjecting it to mass spectrometry, in particular MALDI ToF mass spectrometry using sensitive high mass detection with no digestion or fragmentation step.
The use of the methods of the present invention allows not only a direct mass analysis, but also the determination of the specific binding of a target molecule with its binding ligand, the site(s) of interaction between ligand and target and the relative binding affinity of ligand for the target.
The present application further provides the use of these methods as a very versatile tool in various biological applications such as characterization of antibodies, drug discovery, and complexomics, including automated or higher throughput applications.